The present invention relates to isolated polypeptides having glucanotransferase activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.
4-xcex1-glucanotransferases (EC. 2.4.1.25) catalyzes the reaction of transferring an xcex1-glucan chain from one xcex1-glucan molecule to another (or to glucose). Glucanotransferases are widely distributed in microorganisms (e.g. E. coli) and plant tissues such as potato tubers, germinating barley seeds, sweet potato and spinach.
Recently, glucanotransferases have been found to be capable of catalyzing an intramolecular reaction of an xcex1-glucan. For example, it has been reported that glucanotransferase is capable of catalyzing an intramolecular transglycosylation reaction (cyclization reaction) of amylose, thereby synthesizing cycloamylose having a degree of polymerization (hereinafter xe2x80x9cDPxe2x80x9d) of 17 or more.
Glucanotransferases can be applied for various industrial purposes. As an example, a glucanotransferase can be utilized in processing an xcex1-glucan for the production of a cyclic glucan. In the case for using an enzyme for an industrial purpose, the reaction is desirably conducted at a temperature as high as possible (about 60xc2x0 C. or higher) due to the fact that an xcex1-glucan, i.e. the substrate, is thereby prevented from retrogradation and, at the same time, contamination of the system by microorganism is avoided, or at least reduced.
In general, only glucanotransferases (amylomaltases) having a high activity in a moderate temperature interval (typically from about 30 to 45xc2x0 C.) have been isolated, see, for example, Agric. Biol. Chem. 53, 2653-2659 (1989) and J. Chem. Soc., 44-53 (1956). More recently, a novel heat-resistant glucanotransferase (amylomaltase) also capable of generating a cyclic glucan has been described in EP 0 884 384 A2.
Thus, in a first aspect the present invention relates to an isolated polypeptide having glucanotransferase activity, selected from the group consisting of:
(a) a polypeptide having an amino acid sequence which has at least 65% identity with the amino acid sequence shown as amino acids 1 to 501 of SEQ ID NO:2;
(b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with
(i) a complementary strand of the nucleic acid sequence shown as nucleotides i1 to 1503 of SEQ ID NO:1, or
(ii) a subsequence of (i) of at least 100 nucleotides;
(c) an allelic variant of (a) or (b);
(d) a polypeptide encoded by the glucanotransferase encoding part of the DNA sequence cloned into a plasmid present in Escherichia coli DSM 13049, or a variant thereof having at least 65% identity to said polypeptide; and
(e) a fragment of said polypeptide having glucanotransferase activity.
In a second aspect the present invention relates to an isolated nucleic acid sequence comprising a nucleic acid sequence which encodes for the polypeptide of the invention.
In a third aspect, the present invention relates to an isolated nucleic acid sequence encoding a polypeptide having glucanotransferase activity, selected from the group consisting of:
(a) a nucleic acid sequence having at least 70% identity with the nucleic acid sequence shown as nucleotides 1 to 1503 of SEQ ID NO:1;
(b) a nucleic acid sequence which hybridizes under low stringency conditions with
(i) a complementary strand of the nucleic acid sequence shown as nucleotides 1 to 1503 of SEQ ID NO:1, or
(ii) a subsequence of (i) of at least 100 nucleotides;
(c) an allelic variant of (a) or (b);
(d) the glucanotransferase encoding part of the DNA sequence which has been cloned into a plasmid present in Escherichia coli DSM 13049, or a variant thereof having at least 70% identity to said DNA sequence; and
(e) a subsequence of (a), (b), (c), or (d), wherein the subsequence encodes a polypeptide fragment which has glucanotransferase activity;
or an isolated nucleic acid sequence which is the complementary strand of (a), (b), (c), (d) or (e).
In a fourth aspect the present invention a nucleic acid construct comprising the nucleic acid sequence of the invention operably linked to one or more control sequences capable of directing the expression of the polypeptide in a suitable expression host.
In a fifth aspect the present invention relates to a recombinant expression vector comprising the nucleic acid construct of the invention, a promoter, and transcriptional and translational stop signals.
In a sixth aspect the present invention relates to a recombinant host cell comprising the nucleic acid construct of the invention.
The present invention also relates to methods for producing the polypeptide of the invention; to methods for producing foods and use of the polypeptide of the invention for producing foods as well as to detergent compositions comprising the polypeptide of the invention.